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CHAPTER 29
DNA as Genetic Information
Review site for basics of DNA and genetics:
http://vector.cshl.org/dnaftb
There will be a take-home bonus question today.
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Studies of Heridity
• By geneticists
- describe patterns of inheritance
• traits (phenotypes)
• heritable (passed from parents to offspring)
• cytogeneticists knew that trait inheritance
is associated with the cell nucleus and with
chromosomes
• biochemists knew that chromosomes are
composed of DNA and protein
Q. What is the molecular/biochemical basis of
inheritance?
Parent trait
Offspring trait
How is it known that DNA
contains genetic information
???
Some Important Definitions
• Gene:
- segment of DNA that contains all the
information needed for regulated synthesis
of an RNA or protein product.
• Genome:
- the entire DNA sequence content of an
organism (nuclear DNA)
Biochemical Genetics
Archibald Garrod (1902) - an English doctor
Described “alkaptanurea” disease
Symptom: urine turns black when exposed to air
Found it was due to oxidation of homogentisic acid in urine
homogentisic acid = an intermediate in Phe degradation
(p.897)
Phe
Tyr
homogentisic
acid
Accumulation
of homogentisic
acid
further
metabolites
Biochemical Genetics
Archibald Garrod : important contributions
Described “alkaptanurea” disease
Deduced that it is due to a defective metabolic enzyme
Disease is a hereditary condition (ran in his patients’
families)
Led to concept of “inborn errors of metabolism”
A novel phenotype may reflects a discrete
biochemical difference
Biochemical Genetics
“Real-World Biochemistry”
Aspartame
= a dipeptide:
aspartylphenylalanine
methyl ester
Aspartame is metabolized in the body to its components: aspartic
acid, phenylalanine, and methanol. Like other amino acids, it
provides 4 calories per gram. Since it is about 180 times as sweet as
sugar, the amount of aspartame needed to achieve a given level of
sweetness is less than 1% of the amount of sugar required. Thus
99.4% of the calories can be replaced.
Look on your diet soda cans and read the warning
Biochemical Genetics
Archibald Garrod : important contributions
Proposed that inheritance of a defective metabolic
enzyme leads to inheritance of a phenotype (disease)
Parent trait
defective
enzyme
Offspring trait
George W. Beadle
• born in Wahoo, Ne
• undergraduate degree at UNL
• did graduate work at Cornell
• got a faculty position at CalTech
• ended up as the president of the Univ of Chicago
• did work in the 1930’s & 40’s on Drosophila eyes
and on Neurospora (bread mold)
• “one gene - one enzyme” hypothesis (1941)
• awarded Nobel prize in 1958 (with research
colleagues J. Lederberg and E. Tatum)
George W. Beadle
• Bread Mold: Neurospora crassa
• can grow on minimal media
sucrose
Inorganic salts
biotin
• Beadle selected for nutritional mutants (auxotrophs)
• irradiated fungal spores, grew these up on complete
media, and transferred part of the stock to minimal media
• He looked for mutants that can grow on complete
media but NOT on minimal media
•These mutants are lacking an enzyme for the synthesis
of an essential nutrient
Beadle’s Experiment- Part 1
These mutants may be lacking an enzyme
for the synthesis of an essential nutrient
• Now have nutritional mutants (auxotrophs)
• Figure out which pathway is defective
• By “feeding” experiments
• feed mutants with nutrients (one at a time) to
supplement their defective pathway
•By trial and error, can identify which nutrient the mutant
can’t make, and therefore which pathway is defective
•Beadle did this feeding experiment with amino acids to
look for mutants in amino acid biosynthetic pathways.
Beadle’s Experiment- Part 2
Supplements:
Pro Ser
Arg Lys
Beadle’s Experiment- Part 3
•Using the methods in part1 and part 2, Beadle
generated a collection of Arg auxotrophs
•Then he used feeding experiments to identify
which enzyme was defective in each mutant
Beadle’s Experiment Summary
•Beadle could identify mutants in specific
steps of a pathway
•Assuming each mutant was defective in a
single gene, Beadle postulated that the
different mutant classes each lacked a
different enzyme for Arg biosynthesis
•Therefore, he could show a one-to-one
correspondance between mutation and
absence of an enzyme.
• one gene specifies/encodes one enzyme
Beadle’s experiment gave rise
to a new field called
Biochemical Genetics
Parent
trait
defective
gene
defective
enzyme
Offspring
trait
But it left an open question:
What is the biochemical
nature of a gene?
Frederick Griffith (1928) - a microbiologist
Staphylococcus pneumoniae
Wild-type
mutant
virulent
non-virulent
mutagenized
Smooth colonies
(S strain)
•Produces a polysaccharide coat
- slimy capsule - smooth
•Capsule protects pathogen from being
detected by the host’s immune system.
Inoculate a mouse
Mouse dies and has S strain
in its blood
Rough colonies
(R strain)
•Defective in polysaccharide synthesis no capsule - rough
•No capsule means no protection from
being detected by the host.
Inoculate a mouse
Mouse survives and has no R strain
in its blood
Something from heat-killed S passed into live R
and transformed them into live S.
Griffith called this the “Transforming principle”
Purifying the Transforming Principle (TP)
Avery, McCarty and McLeod - Biochemists (1944)
Heat-killed S bacteria
Mix with live R
TP is probably not a protein
(heat treatment!)
Cell-free extract
Mix with live R
Very pure preparation of DNA
Mouse dies and has live S
in its blood
Mouse dies and has live S
in its blood
Mix with live R
Add a protease
Add a nuclease
Still transforms!
Lose transforming activity
Mouse dies and has live S
in its blood
It’s DNA!!!
Further Proof Required
• Good evidence that DNA is the
transforming principle
• There is no proof yet that the DNA itself
is stably inherited
• i.e. no proof that transforming DNA from
dead S actually gets inside live R to turn
it into S.
Further Proof Provided
By virologists (1952)
Alfred Hershey
Martha Chase
Hershey and Chase clearly linked DNA and heridity
Bacteriophage - virus infects
bacteria
•Bacteriophage attach to a host
cell via cell surface receptors
• inject their DNA
•Host cell now makes viral protein
and more DNA
•New virus assembles inside the
cel
•Cells lyse and phage are
released.
Hershey and Chase prepared radioactivelylabeled bacteriophage
DNA
rest of
virus is
protein
1) to make virus with labeled DNA
Mix: host bacteria
virus
32P (radioactive)
Progeny viruses have radioactive DNA
2) to make virus with labeled protein
Mix: host bacteria
virus
35S (radioactive)
Progeny viruses have radioactive
protein
Take these viruses and do a new infection to see
what gets injected into a host cell
Further Proof Provided
• In 1952, Hershey and Chase, studying
bacteriophages, labelled DNA with 32P
and protein with 35S
• Bacteriophage progeny produced by
infection of bacteria contained 32P (thus
DNA from the original phage), but not
35S (from the protein)!
DNA Structure Model 1953
You should read Watson and Crick’s original
Nature paper at the following site:
http://www.nature.com/genomics/human/
(Scroll to the bottom of the page and click on “full text”
in the green Watson and Crick box)
DNA Structure Model 1953
WATSON, J. D. & CRICK, F. H. C.
Medical Research Council Unit for the Study of Molecular Structure of Biological
Systems, Cavendish Laboratory,Cambridge.
A Structure for Deoxyribose Nucleic Acid
We wish to suggest a structure for the salt of deoxyribose nucleic
acid (D.N.A.). This structure has novel features which are of
considerable biological interest. …..
……… It has not escaped our notice that the specific pairing we
have postulated immediately suggests a possible copying
mechanism for the genetic material.
The Human Genome Project
What is the Human
Genome Project?
• U.S. govt. project coordinated by the Department
of Energy and the National Institutes of Health
• goals (1998-2003)
– identify the approximate 100,000 genes in human DNA
– determine the sequences of the 3 billion bases that
make up human DNA
– store this information in databases
– develop tools for data analysis
– address the ethical, legal, and social issues that arise
from genome research
Why is the Department of
Energy involved?
-after atomic bombs were dropped during War
War II, Congress told DOE to conduct studies to
understand the biological and health effects of
radiation and chemical by-products of all energy
production
-best way to study these effects is at the DNA
level
Whose genome is being
sequenced?
• the first reference genome is a composite
genome from several different people
• generated from 10-20 primary samples
taken from numerous anonymous donors
across racial and ethnic groups
Benefits of HGP Research
• improvements in medicine
• microbial genome research for fuel and
environmental cleanup
• DNA forensics
• improved agriculture and livestock
• better understanding of evolution and
human migration
• more accurate risk assessment
Ethical, Legal, and Social
Implications of HGP Research
•
•
•
•
•
•
•
•
fairness in the use of genetic information
privacy and confidentiality
psychological impact and stigmatization
genetic testing
reproductive issues
education, standards, and quality control
commercialization
conceptual and philosophical implications
For More Information...
Human Genome Project Information Website
http://www.ornl.gov/hgmis
Insights Learned from the
Sequence
• What has been learned from analysis
of the working draft sequence of the
human genome? What is still
unknown?
(information taken from Science,
Nature, Wellcome Trust, and Human
Genome News)
By the Numbers
• The human genome contains 3164.7 million nucleotide bases
(A, C, T, and G).
• The average gene consists of 3000 bases, but sizes vary
greatly, with the largest known human gene being dystrophin
(2.4 million bases).
• The total number of genes is estimated at 30,000 to 35,000,
much lower than previous estimates of 80,000 to 140,000 that
had been based on extrapolations from gene-rich areas as
opposed to a composite of gene-rich and gene-poor areas.
• The order of almost all (99.9%) nucleotide bases are exactly
the same in all people.
•The functions are unknown for over 50% of discovered genes.
The Wheat from the Chaff
• Less than 2% of the genome encodes for the production of
proteins.
• Repeated sequences that do not code for proteins ("junk DNA")
make up at least 50% of the human genome.
• Repetitive sequences are thought to have no direct functions,
but they shed light on chromosome structure and dynamics. Over
time, these repeats reshape the genome by rearranging it,
thereby creating entirely new genes or modifying and reshuffling
existing genes.
How It's Arranged
• The human genome's gene-dense "urban centers" are
predominantly composed of the DNA building blocks G and C.
• In contrast, the gene-poor "deserts" are rich in the DNA building
blocks A and T. GC- and AT-rich regions usually can be seen
through a microscope as light and dark bands on chromosomes.
• Genes appear to be concentrated in random areas along the
genome, with vast expanses of non-coding DNA between.
• Stretches of up to 30,000 C and G bases repeating over and
over often occur adjacent to gene-rich areas, forming a barrier
between the genes and the "junk DNA." These CpG islands are
believed to help regulate gene activity.
How do we Compare to Other
Organisms
• Humans have on average three times as many kinds of
proteins as the fly or worm because of mRNA transcript
"alternative splicing" and chemical modifications to the proteins.
This process can yield different protein products from the same
gene.
• Humans share most of the same protein families with worms,
flies, and plants, but the number of gene family members has
expanded in humans, especially in proteins involved in
development and immunity.
Variations and Mutations
• Scientists have identified about 1.4 million locations where
single-base DNA differences (SNPs) occur in humans. This
information promises to revolutionize the processes of finding
chromosomal locations for disease-associated sequences and
tracing human history.
• The ratio of germline (sperm or egg cell) mutations is 2:1 in
males vs females. Researchers point to several reasons for
the higher mutation rate in the male germline, including the
greater number of cell divisions required for sperm formation
than for eggs.
Impact
The draft sequence already is having an impact on finding
genes associated with disease.
Over 30 genes have been pinpointed and associated with
breast cancer, muscle disease, deafness, and blindness.
Additionally, finding the DNA sequences underlying such
common diseases as cardiovascular disease, diabetes,
arthritis, and cancers is being aided by the human variation
maps (SNPs).
These genes and SNPs provide focused targets for the
development of effective new therapies.
There will be a take-home bonus question today.